replication 13

11

DNA STRUCTURE DNA STRUCTURE AND AND

REPLICATIONREPLICATION

DR.K.S.SODHI PROFESSOR MMIMS&R

22

Central Dogma

RNA

DNA

Protein

轉錄

轉譯

Transcription

Translation

Replication複製

逆轉錄 ReverseTranscription

Juang RH (2004) BCbasics

33

ARTHUR KORNBERG1958ARTHUR KORNBERG1958

44

23 paternaldirectories

23 maternalequivalents

Total 35,000 files

Replication

Nucleus23 x 2

In 46 chromosomes

Homologous chromosomes

Before celldivision

3,000 MB

Cell

Nucleus


55

Site-directed mutagenesis

CAG

GTC

CAG

GCC

CAG

GCC

CAG

+ polymerase

+ primer

replication

GCC

CGGMutant

Thr

translation

Wild type

GTC

CAG

Val

translation

Only one amino acid changed

Wild type protein

Mutant protein

primer

(1)

(2)

(3)

(5)

(4)

(6)

Val → ThrSmith (1993) Jua

ng

RH

(2

00

4)

BC

ba

sics

66

AbbreviationsAbbreviations dsDNA.dsDNA. ssDNA.ssDNA. Ori.Ori. SSBsSSBs DnaK,dnaJ,dnaEDnaK,dnaJ,dnaE DNA Helicase.DNA Helicase. dRibosedRibose ApoptosisApoptosis Leading & Laging strands.Leading & Laging strands. Gyrase.Gyrase.

Speed:100nts/sec. total 9 hours to Speed:100nts/sec. total 9 hours to complete in a typical cell.complete in a typical cell.

Double stranded DNADouble stranded DNA Single stranded DNASingle stranded DNA Origin of replication.Origin of replication. Single strand Binding.Single strand Binding. Heat shock proteins EHeat shock proteins E Unwind short segmenUnwind short segmen De-OxyriboseDe-Oxyribose Programmed cell deathProgrammed cell death One strech, multiple streches.One strech, multiple streches. Negative supercoiling using ATPNegative supercoiling using ATP

77

cDNA asingle stranded DNA molecule cDNA asingle stranded DNA molecule that is complementary to mRNA and is that is complementary to mRNA and is synthesised from it by the action of synthesised from it by the action of reverse transcriptase.reverse transcriptase.

miRNAS micro RNAs 21-25 nucleotide miRNAS micro RNAs 21-25 nucleotide long.long.

Sines Short interspread repeat Sines Short interspread repeat sequences.sequences.

Si RNA silencing RNA 21-25 nt length Si RNA silencing RNA 21-25 nt length can cause gene knockdown. can cause gene knockdown.

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PROTEINS & FUNCTIONSPROTEINS & FUNCTIONS

DNA polymerasesDNA polymerases Helicases.Helicases. Topoisomerases.Topoisomerases. DNA primaseDNA primase

SSB proteinsSSB proteins

DNA LigaseDNA Ligase

Polymerisation.Polymerisation. Unwinding of DNA.Unwinding of DNA. Remove supercoiling.Remove supercoiling. Initiates synth. of RNA Initiates synth. of RNA

primer.primer. Prevent reanealing of Prevent reanealing of

dsDNA.dsDNA. Seals the nick in Seals the nick in

okazaki fragments.okazaki fragments.

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REQUIREMENTSREQUIREMENTS

Four activated precursors of Four activated precursors of dATP,dGTP,dCTP andTTP MgdATP,dGTP,dCTP andTTP Mg++++ . .

Template Strand.Template Strand. Primer with free3’-OH group(10-200)Primer with free3’-OH group(10-200) Elongation proceeds 5’—3’ direction.Elongation proceeds 5’—3’ direction. Removal of mismatched nucleotides.Removal of mismatched nucleotides. Error rate is less than 10 Error rate is less than 10 -8 -8 per bp.

3’ 3’ hydroxyl group attack(nucleophillic) on po4 of recently attached nucleotide.

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DNADNA DNA stands for deoxyribose nucleic acid This chemical substance is present in the

nucleus of all cells in all living organisms DNA controls all the chemical changes

which take place in cells The kind of cell which is formed, (muscle,

blood, nerve etc) is controlled by DNA The kind of organism which is produced

(buttercup, giraffe, herring, human etc) is controlled by DNA The kind of organism which is produced (buttercup, giraffe, herring, human etc) is controlled by DNA

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Ribose is a sugar, like glucose, but with only fivecarbon atoms in its molecule

Deoxyribose is almost the same but lacks one oxygen atom

Both molecules may be represented by the symbol

Ribose & deoxyriboseRibose & deoxyribose

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The most common organic bases are

Adenine (A)

Thymine (T)

Cytosine (C)

Guanine (G)

The basesThe bases

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The deoxyribose, the phosphate and one of the bases

adenine

deoxyribose

PO4

Combine to form a nucleotide

NucleotidesNucleotides

1414

A molecule of DNA is formed by

millions of nucleotides joined together in a long

chain

PO4

PO4

PO4

PO4

sugar-phosphate backbone + bases

Joined nucleotidesJoined nucleotides

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PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

2-stranded DNA2-stranded DNA

1616

REPLICATION STEPSREPLICATION STEPS

A. INITIATIONA. INITIATION B. ELONGATION.B. ELONGATION. C. TERMINATION.C. TERMINATION.

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DNA ReplicationDNA Replication

Priming:Priming:

1.1. RNA primersRNA primers: before new DNA strands can : before new DNA strands can form, there must be small pre-existing form, there must be small pre-existing primers (RNA) primers (RNA) present to start the addition of present to start the addition of new nucleotides new nucleotides (DNA Polymerase)(DNA Polymerase)..

2.2. PrimasePrimase: enzyme that polymerizes : enzyme that polymerizes (synthesizes) the (synthesizes) the RNA PrimerRNA Primer..

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This DNA polymerase replaces the RNA primer with DNA. This is a different type of DNA polymerase from the main DNA polymerase which synthesises DNA on a DNA template.

Another DNA polymerase:

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In E. coli the main enzyme is DNA polymerase III .And the enzyme that replaces the RNA primer with DNA is DNA polymerase I. When the RNA primer has been replaced with DNA, there is a gap between the two Okazaki fragments and this is sealed by DNA ligase.

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DNA ligase seals the gap left between Okazaki fragments after the primer is removed. As the Okazaki fragments are joined, the new lagging strand becomes longer and longer.

DNA ligase:

Location: At the replication fork. Function: Unwinds the DNA double helix.

Helicase:

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Location: On the template strands.

Function: Synthesizes new DNA in the 5' to 3' direction using the base information on the template strand to specify the nucleotide to insert on the new chain. Also does some proofreading; that is, it checks that the new nucleotide being added to the chain carries the correct base as specified by the template DNA.

DNA polymerase:

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The new DNA strand made discontinuously in the direction opposite to the direction in which the replication fork is moving.

The new DNA strand made continuously in the same direction as movement of the replication fork.

Lagging Strand:

Leading strand:

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If an incorrect base pair is formed, DNA polymerase can delete the new nucleotide and try again. In E. coli the enzyme used for all new DNA synthesis except for the replacement of the RNA primers is DNA polymerase III. DNA polymerase I replaces the primers.

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Location: On the template strand which dictates new DNA synthesis away from the direction of replication fork movement.

Okazaki fragment:

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Function: A building block for DNA synthesis of the lagging strand. On one template strand, DNA polymerase synthesizes new DNA in a direction away from the replication fork movement. Because of this, the new DNA synthesized on that template is made in a discontinuous fashion; each segment is called an Okazaki fragment.

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Location: Wherever the synthesis of a new DNA fragment is to commence. Function: DNA polymerase cannot start the synthesis of a new DNA chain, it can only extend a nucleotide chain primer. Primase synthesizes a short RNA chain that is used as the primer for DNA synthesis by DNA polymerase.

Primase:

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Location: On single-stranded DNA near the replication fork. Function: Binds to single-stranded DNA to make it stable.

Single-strand binding (SSB) proteins

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Alternative models of DNA replication

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DNA Replication 1

Models of DNA replication: -Meselson-Stahl Experiment

DNA synthesis and elongation

DNA polymerases

Origin and initiation of DNA replication

Prokaryote/eukaryote models

Telomere replication

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Let us animate the Let us animate the process of replicationprocess of replication

3232

H bonds break Two strands seperate

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Sugar phosphate backbone is made by joining the adjcent nucleotides ( DNA polymarase enzyme( ) )

Nucleotides with Complementary bases are assembled alongside each strands

3434

Two identical DNA molecules are formed

3535

1958: Matthew Meselson & Frank Stahl’s Experiment

Semiconservative model of DNA replication (Fig. 3.2)

3636

1955: Arthur Kornberg

Worked with E. coli. Discovered the mechanisms of DNA synthesis.

Four components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)

2. DNA template

3. DNA polymerase (Kornberg enzyme)

4. Mg 2+ (optimizes DNA polymerase activity)

1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)

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Three main features of the DNA synthesis reaction:

1. DNA polymerase I catalyzes formation of phosphodiester bondbetween 3’-OH of the deoxyribose (on the last nucleotide) and the 5’-phosphate of the dNTP.

• Energy for this reaction is derived from the release of two of the three phosphates.

2. DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening process.

• rate ≤ 800 dNTPs/second• low error rate

3. Direction of synthesis is 5’ to 3’

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DNA elongation

3939

DNA elongation (Fig. 3.3a):

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There are many different types of DNA polymerase

Polymerase Polymerization (5’-3’)

Exonuclease (3’-5’)

Exonuclease (5’-3’)

#Copies

I YES YES YES 400

II YES NO YES?

III YES YES YES20-40

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3’ to 5’ exonuclease activity = ability to remove nucleotides from the 3’ end of the chain

Important proofreading ability – Without proofreading error rate (mutation rate) is 1

x 10-6

– With proofreading error rate is 1 x 10-9 (1000-fold decrease)

5’ to 3’ exonuclease activity functions in DNA replication & repair.

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Eukaryotic enzymes:

Five DNA polymerases from mammals.

Polymerase (alpha): nuclear, DNA replication, no proofreading

Polymerase (beta): nuclear, DNA repair, no proofreading

Polymerase (gamma): mitochondria, DNA repl., proofreading

Polymerase (delta): nuclear, DNA replication, proofreading

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Polymerase (epsilon): nuclear, DNA repair (?), proofreading

Different polymerases for nucleus and mtDNA

Some polymerases proofread; others do not.

Some polymerases used for replication; others for repair

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Origin of replication (e.g., the prokaryote example):

Begins with double-helix denaturing into single-strands thus exposing the bases.

Exposes a replication bubble from which replication proceeds in both directions.

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Initiation of replication, major elements:

Segments of single-stranded DNA are called template strands.

Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.

Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate).(Hydrolysis of ATP causes a shape change in DNA helicase)

DNA primase next binds to helicase producing a complex called a primosome (primase is required for synthesis),

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Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides.

Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA primer.

The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with DNA ligase.

Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded template DNA during the process.

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DNA replication is continuous on the leading strand and semidiscontinuous on the lagging strand:

Unwinding of any single DNA replication fork proceeds in one direction.

The two DNA strands are of opposite polarity, and DNA polymerases only synthesize DNA 5’ to 3’.

Solution: DNA is made in opposite directions on each template.

•Leading strand synthesized 5’ to 3’ in the direction of the replication fork movement.

continuous

requires a single RNA primer

•Lagging strand synthesized 5’ to 3’ in the opposite direction.

semidiscontinuous (i.e., not continuous)

requires many RNA primers

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3

Polymerase III

5’ 3

’

Leading strand

base pairs

5’

5’

3’

3’

Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

Helicase +

Initiator Proteins

ATP

SSB Proteins

RNA Primer

primase

2Polymerase III

Lagging strand

Okazaki Fragments

1

RNA primer replaced by polymerase I& gap is sealed by ligase

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Two Libraries ： cDNA Library vs Genomic Library

mRNA

cDNA

Reverse transcription

Chromosomal DNA

Restriction digestion

Genes in expression Total Gene

Complete gene Gene fragments

SmallerLibrary

Larger Library

Vector:Plasmid or phageVector: Plasmid


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Restriction Mapping of DNA

A B 10 kb

8 kb2 kb

A

7 kb3 kb

B

5 kb3 kb2 kb

A+B

CK A B A+B M

Restriction enzymes


5454

The Specific Cutting and Ligation of DNA

GAATTC

CTTAAG

GAATTC

CTTAAG

G

CTTAA

AATTC

G

AATTC

G

G

CTTAA

G

CTTAA

AATTC

G

G

CTTAA

AATTC

G

G

CTTAA

AATTC

G

EcoRI

DNA LigaseEcoRI sticky end EcoRI sticky end


5656

DNA ligase seals the gaps between Okazaki fragments with aphosphodiester bond (Fig. 3.7)TIME: E.coli 30 minutes,Humans: 24 Hours.

5757Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 3.5 - Model of DNA replication


Fig. 3.5 - Model of DNA replication

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Concepts and terms to understand:

Why are gyrase and helicase required?

The difference between a template and a primer?

The difference between primase and polymerase?

What is a replication fork and how many are there?

Why are single-stranded binding (SSB) proteins required?

How does synthesis differ on leading strand and lagging strand?

Which is continuous and semi-discontinuous?

What are Okazaki fragments?

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Replication of circular DNA in

E. coli (3.10):

1. Two replication forks result in a theta-like () structure.

2. As strands separate, positive supercoils form elsewhere in the molecule.

3. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.

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Rolling circle model of DNA replication (3.11):

1. Common in several bacteriophages including .

2. Begins with a nick at the origin of replication.

3. 5’ end of the molecule is displaced and acts as primer for DNA synthesis.

4. Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly).

5. During viral assembly the DNA is cut into individual viral chromosomes.

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DNA replication in eukaryotes:

Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges:

Major checkpoints in the system

1. Cells must be large enough, and the environment favorable.

2. Cell will not enter the mitotic phase unless all the DNA has replicated.

3. Chromosomes also must be attached to the mitotic spindle for mitosis to complete.

4. Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks).

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• Each eukaryotic chromosome is one linear DNA double helix

• Average ~108 base pairs long

• With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days.

• Solution ---> DNA replication initiates at many different sites simultaneously.

Fig. 3.14

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Fig. 3.13 - Replication forks visible in Drosophila

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(or telomeres What about the ends ) of linear chromosomes?

DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. this gap is not filled, chromosomes would become shorter each round of replication!

Solution:

1. Eukaryotes have tandemly repeated sequences at the ends of their chromosomes.

2. Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of of new repeats.

3. Compensates by lengthening the chromosome.

4. Absence or mutation of telomerase activity results in chromosome shortening and limited cell division.


Fig. 3.16 Synthesis of telomeric DNA by telomerase

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Final Step - Assembly into Nucleosomes:

• As DNA unwinds, nucleosomes must disassemble.

• Histones and the associated chromatin proteins must be duplicated by new protein synthesis.

• Newly replicated DNA is assembled into nucleosomes almost immediately.

• Histone chaperone proteins control the assembly.

Fig. 3.17

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DNA DAMAGE DNA DAMAGE AND REPAIRAND REPAIR

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DNA DAMAGE

1. SPONTANEOUS2. ENVIRONMENTAL

AGENTS3. REPLICATION

PHYSICAL

CHEMICAL

BIOLOGICAL

7070

Mis-incorporation

of bases

Chemicals UV-radiation

X-radiation

SpontaneousDe-amination

of bases

CAUSES OF

DAMAGE

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SingleBase

alterations

CrossLinkage

Chainbreaks

TwoBase

alterations

TYPESOF

DAMAGE

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1. 1. SINGLE BASE ALTERATIONSSINGLE BASE ALTERATIONS

DEPURINATIONDEPURINATION DEAMINATION OF CYTOSINE TO URACILDEAMINATION OF CYTOSINE TO URACIL DEAMINATION OF ADENINE TO DEAMINATION OF ADENINE TO

HYPOXANTHINEHYPOXANTHINE ALKYLATION OF BASESALKYLATION OF BASES INSERTION OR DELETION OF NUCLEOTIDESINSERTION OR DELETION OF NUCLEOTIDES BASE ANALOG INCORPORATIONBASE ANALOG INCORPORATION

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2. 2. TWO BASE ALTERATIONSTWO BASE ALTERATIONS

UV INDUCED THYMINE-THYMINE UV INDUCED THYMINE-THYMINE DIMERSDIMERS

BIFUNCTIONAL ALKYLATING AGENTSBIFUNCTIONAL ALKYLATING AGENTS

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3. 3. CHAIN BREAKSCHAIN BREAKS

IONIZING RADIATION INDUCEDIONIZING RADIATION INDUCED RADIOACTIVE DISINTEGRATION OF RADIOACTIVE DISINTEGRATION OF

BACKBONE ELEMENTBACKBONE ELEMENT FREE-RADICAL INDUCEDFREE-RADICAL INDUCED

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4. CROSS LINKAGE4. CROSS LINKAGE

BETWEEN BASES IN SAME AND BETWEEN BASES IN SAME AND OPPOSITE STRANDOPPOSITE STRAND

BETWEEN DNA AND PROTEIN BETWEEN DNA AND PROTEIN MOLECULESMOLECULES

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1. Proofreading

and editing

2. MismatchRepairsystem

3. BaseExcision

repair

4. NucleotideExcisionRepair

5. Photo-Reactivation

OrDirectrepair

6. DoubleStrandBreakRepair

7.Transcription-Coupled

repair

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1. PROOF READING AND 1. PROOF READING AND EDITINGEDITING Despite double Despite double

monitoring during monitoring during replication, first at time of replication, first at time of incorporation of bases and incorporation of bases and second by later follow up second by later follow up energy requiring energy requiring processes, some processes, some mispaired bases persist mispaired bases persist which have to be removed which have to be removed by other enzyme systems.by other enzyme systems.

The proof reading activity The proof reading activity is carried out by 3’-5’ is carried out by 3’-5’ exonuclease activities of exonuclease activities of DNA polymerase III and DNA polymerase III and I.I.

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2. MISMATCH REPAIR SYSTEM2. MISMATCH REPAIR SYSTEM

This mechanism operates immediately after This mechanism operates immediately after DNA replication.DNA replication.

Sometimes the replication errors escape the Sometimes the replication errors escape the DNA proofreading function. This mechanism DNA proofreading function. This mechanism checks for the correction of escaped bases.checks for the correction of escaped bases.

Specific proteins scan the newly synthesized Specific proteins scan the newly synthesized DNA by the following mechanisms:DNA by the following mechanisms:

1.1. Identification of mismatched strandIdentification of mismatched strand2.2. Repair of mispaired base.Repair of mispaired base.

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In error detection, In error detection, parent strand is parent strand is identified first with the identified first with the help of GATC-help of GATC-sequences, that occur sequences, that occur approx. once after every approx. once after every thousand nucleotides.thousand nucleotides.

It is methylated at It is methylated at adenine residue.adenine residue.

The methylation does The methylation does not occur immediately not occur immediately after replication. So, the after replication. So, the new strand is not new strand is not methylated and is easily methylated and is easily identified.identified.

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Secondly, on the new strand, GATC-endonuclease Secondly, on the new strand, GATC-endonuclease ‘nicks’ the mismatched strand.‘nicks’ the mismatched strand.

This faulty strand is digested by exonuclease.This faulty strand is digested by exonuclease. An extensive region, from the mismatched area till An extensive region, from the mismatched area till

the next GATC-sequence is removed.the next GATC-sequence is removed. This gap is filled by the DNA polymerase I, in 5’-3’ This gap is filled by the DNA polymerase I, in 5’-3’

direction.direction. Clinical significance-Clinical significance- A defect in mismatch repair A defect in mismatch repair

in humans has been known to cause hereditary non-in humans has been known to cause hereditary non-polyposis colon cancer (HNPCC).polyposis colon cancer (HNPCC).

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3. BASE EXCISION REPAIR3. BASE EXCISION REPAIR This mechanism operates all the time in the cells.This mechanism operates all the time in the cells. The bases of DNA can be altered:The bases of DNA can be altered:a)a) Spontaneously Spontaneously -- cytosine uracil. cytosine uracil.b)b) Deaminating compounds -Deaminating compounds - like NO, which is formed like NO, which is formed

from nitrosamines,nitrites, and nitrates.from nitrosamines,nitrites, and nitrates. - NO is a potent de-aminating compound, that converts:- NO is a potent de-aminating compound, that converts:i.i. Ctytosine uracilCtytosine uracilii.ii. Adenine hypoxanthineAdenine hypoxanthineiii.iii. Guanine xanthineGuanine xanthinec)c) Bases can also be lost spontaneously - Bases can also be lost spontaneously - approximately, approximately,

10,000 purine bases are lost spontaneously per cell per 10,000 purine bases are lost spontaneously per cell per day.day.

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Following mechanisms Following mechanisms operate to correct such base operate to correct such base alterations or losses:alterations or losses:

1.1. Removal of abnormal Removal of abnormal bases-bases- Abnormal bases are Abnormal bases are recognized by specific recognized by specific glycosylasesglycosylases. .

-they hydrolytically cleave -they hydrolytically cleave them from deoxy-ribose-them from deoxy-ribose-phosphate backbone of the phosphate backbone of the strand.strand.

-this results in either A-this results in either Apurinic purinic or Apyrimidinicor Apyrimidinic site, site, referred to as referred to as AP- siteAP- site..

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2.2. Repair of AP-site -Repair of AP-site - AP-endonucleaseAP-endonuclease recognizes the recognizes the empty site and starts excision by making a cut at 5’-end of empty site and starts excision by making a cut at 5’-end of AP-site.AP-site.

- - deoxy-ribose-phosphate lyasedeoxy-ribose-phosphate lyase removes the single, empty, removes the single, empty, sugar-phosphate residue and gap is finally filled by sugar-phosphate residue and gap is finally filled by DNA DNA polymerase Ipolymerase I and nick is sealed by and nick is sealed by DNA ligaseDNA ligase..

NOTE..NOTE.. By the similar series of steps involving initially the By the similar series of steps involving initially the

recognition of the defect, the alkylated bases and base recognition of the defect, the alkylated bases and base analogs can be removed from DNA. And thus, DNA analogs can be removed from DNA. And thus, DNA returns to its original information content.returns to its original information content.

This mechanism is efficient only for replacement of a This mechanism is efficient only for replacement of a single base but is not efficient for replacing regions of single base but is not efficient for replacing regions of damaged DNA.damaged DNA.

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Recognition and excision of Recognition and excision of defectdefect

(eg:-UV induced dimers)(eg:-UV induced dimers) First,a UV-specific First,a UV-specific

endonuclease recognizes the endonuclease recognizes the dimer and cleaves the dimer and cleaves the damaged strand at damaged strand at phosphodiester bonds on both phosphodiester bonds on both 5’ side and 3’ side of the 5’ side and 3’ side of the dimer.dimer.

The damaged oligonucleotide The damaged oligonucleotide is released, leaving a gap in is released, leaving a gap in the DNA strand that formerly the DNA strand that formerly contained the dimer.contained the dimer.

This gap is filled by This gap is filled by DNA DNA polymerase Ipolymerase I and nick is and nick is sealed by sealed by DNA ligaseDNA ligase..

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5. PHOTO-REACTIVATION OR 5. PHOTO-REACTIVATION OR DIRECT REPAIRDIRECT REPAIR

This is also called ‘This is also called ‘light light induced repair’.induced repair’.

The enzyme photo-The enzyme photo-reactivating enzyme (PR reactivating enzyme (PR enzyme) brings about an enzyme) brings about an enzymatic cleavage of enzymatic cleavage of thymine dimers activated by thymine dimers activated by the visible lightthe visible light

It leads to restoration of It leads to restoration of monomeric condition.monomeric condition.

Co-enzymes required for the Co-enzymes required for the reaction are FADH2 and reaction are FADH2 and THF.THF.

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6. DOUBLE STRAND BREAK 6. DOUBLE STRAND BREAK REPAIRREPAIR High energy radiation, oxidative free radicals or some High energy radiation, oxidative free radicals or some

chemotherapeutic agents bring about double-stranded chemotherapeutic agents bring about double-stranded breaks in DNA, or may also occur naturally during breaks in DNA, or may also occur naturally during naturally during gene rearrangements.naturally during gene rearrangements.

They are potentially lethal to the cell.They are potentially lethal to the cell. They cannot be repaired by excising single strand and They cannot be repaired by excising single strand and

using the other strand as template to replace missing using the other strand as template to replace missing nucleotides.nucleotides.

It is repaired by 2 ways:It is repaired by 2 ways:

1.1. Non-homologous end-joining repair.Non-homologous end-joining repair.

2.2. Homologous recombination repair.Homologous recombination repair.

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1.1. Non-homologous end joining repair-Non-homologous end joining repair- In this system the two ends of DNA are In this system the two ends of DNA are

brought together by a group of proteins and brought together by a group of proteins and thereby the ends are re-ligated.thereby the ends are re-ligated.

This system does not require that the 2 DNA This system does not require that the 2 DNA sequences have any homology.sequences have any homology.

2.2. Homologous recombination repair-Homologous recombination repair- This system uses the enzymes that normally This system uses the enzymes that normally

perform genetic recombination between perform genetic recombination between homologous chromosomes during meiosis.homologous chromosomes during meiosis.

This is called sister-strand exchange.This is called sister-strand exchange.

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7. TRANSCRIPTION COUPLED 7. TRANSCRIPTION COUPLED REPAIRREPAIR

When RNA polymerase transcribes a When RNA polymerase transcribes a gene, as it encounters a damaged gene, as it encounters a damaged region, the transcription stops.region, the transcription stops.

The excision repair enzymes repair The excision repair enzymes repair the area and then transcription the area and then transcription resumes.resumes.

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CLINICALCLINICAL

DISORDERSDISORDERS

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1. 1. XERODERMA XERODERMA PIGMENTOSAPIGMENTOSA

Autosomal recessive in nature.Autosomal recessive in nature. UV- specific exonuclease is deficient.UV- specific exonuclease is deficient. Cutaneous hypersensitivity to UV-rays.Cutaneous hypersensitivity to UV-rays. Blisters on skin.Blisters on skin. Hyperpigmentation.Hyperpigmentation. Corneal ulcer.Corneal ulcer. Death occurs due to formation of cancers of Death occurs due to formation of cancers of

skin.skin.

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2. ATAXIA TELANGIECTASIA2. ATAXIA TELANGIECTASIA

Autosomal recessive in Autosomal recessive in nature.nature.

Increased sensitivity to X-Increased sensitivity to X-rays and UV-rays.rays and UV-rays.

Progressive cerebellar Progressive cerebellar ataxia.ataxia.

Oculo-cutaneous Oculo-cutaneous telangiectasia.telangiectasia.

Frequent sino-pulmonary Frequent sino-pulmonary infections.infections.

Lympho-reticular neoplasm.Lympho-reticular neoplasm.

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3. BLOOM’S SYNDROME3. BLOOM’S SYNDROME

Chromosomal breaks or Chromosomal breaks or rearrangements are seen.rearrangements are seen.

Defect lies in DNA helicase or Defect lies in DNA helicase or ligase.ligase.

Facial erythmia.Facial erythmia. Photosensitivity.Photosensitivity. Lympho-reticular Lympho-reticular

malignancies.malignancies.

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4.FANCONI SYNDROME4.FANCONI SYNDROME

Lethal aplastic anaemia,due to Lethal aplastic anaemia,due to defective DNA repair.defective DNA repair.

Cells can not repair interstrand cross-Cells can not repair interstrand cross-links, or damage induced by X-Rays.links, or damage induced by X-Rays.

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5.Cockayne’sSyndrome 5.Cockayne’sSyndrome &Retinoblastoma&Retinoblastoma

Defects in DNA repair.Defects in DNA repair. Cells from patients with some Cells from patients with some

chromosomal abnormalities eg Down chromosomal abnormalities eg Down Syndrome may also show aberrant Syndrome may also show aberrant DNA repair.DNA repair.

9696

INHIBITOR OF DNA INHIBITOR OF DNA REPLICATIONREPLICATION

Anthracyclines cause chain Anthracyclines cause chain breakageSu.bstances that act breakageSu.bstances that act directly on DNA Polymerases eg. directly on DNA Polymerases eg. Acyclovir inhibits the DNA Acyclovir inhibits the DNA polymerase of herpes simplex.polymerase of herpes simplex.

2’-dideoxyazidocytidine is a inhibitor 2’-dideoxyazidocytidine is a inhibitor of bacterial primase,and of bacterial primase,and cournermycin,novobiocin,oxolinic cournermycin,novobiocin,oxolinic acid and nalidixic acid are effective acid and nalidixic acid are effective inhibitor of DNA gyrase in bacteria.inhibitor of DNA gyrase in bacteria.

9797

TOPOISOMERASE I TOPOISOMERASE I INHIBITORINHIBITOR

Topoisomerase is essential for DNA Topoisomerase is essential for DNA replication and cell growth.replication and cell growth.

Certain drugs produces double strand Certain drugs produces double strand breaks in DNA that are irreversible and breaks in DNA that are irreversible and can lead to cell death.can lead to cell death.

Eg. Quilnolne antibiotics,anthracyclines Eg. Quilnolne antibiotics,anthracyclines active for treatment of lung, ovarian and active for treatment of lung, ovarian and colorectal cancer.The Camptothecins were colorectal cancer.The Camptothecins were discovered from extract of tree discovered from extract of tree Camptotheca acuminata.Camptotheca acuminata.

9898

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